Method of transferring at least two saccharide units with a polyglycosyltransferase

ABSTRACT

The present invention relates to a method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and a gene encoding such a polyglycosyltransferase.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method of transferring atleast two saccharide units with a polyglycosyltransferase, apolyglycosyltransferase and a gene encoding such apolyglycosyltransferase.

[0003] 2. Discussion of the Background:

[0004] Biosynthesis of Oligosaccharides

[0005] Oligosaccharides are polymers of varying number of residues,linkages, and subunits. The basic subunit is a carbohydratemonosaccharide or sugar, such as mannose, glucose, galactose,N-acetylglucosamine, N-acetylgalactosamine, and the like. The number ofdifferent possible stereoisomeric oligosaccharide chains is enormous.

[0006] Oligosaccharides and polysaccharides play an important role inprotein function and activity, by serving as half-life modulators, and,in some instances, by providing structure. As pointed out above,oligosaccharides are critical to the antigenic variability, and henceimmune evasion, of Neisseria, especially gonococcus.

[0007] Numerous classical techniques for the synthesis of carbohydrateshave been developed, but these techniques suffer the difficulty ofrequiring selective protection and deprotection. Organic synthesis ofoligosaccharides is further hampered by the liability of many glycosidicbonds, difficulties in achieving regioselective sugar coupling, andgenerally low synthetic yields. In short, unlike the experience withpeptide synthesis, traditional synthetic organic chemistry cannotprovide for quantitative, reliable synthesis of even fairly simpleoligosaccharides.

[0008] Recent advances in oligosaccharide synthesis have occurred withthe isolation of glycosyltransferases from natural sources. Theseenzymes can be used in vitro to prepare oligosaccharides andpolysaccharides (see, e.g., Roth, U.S. Pat. No. 5,180,674). Theadvantage of biosynthesis with glycosyltransferases is that theglycosidic linkages formed by enzymes are highly stereo andregiospecific. However, each enzyme catalyzes linkage of specific sugardonor residues to other specific acceptor molecules, e.g., anoligosaccharide or lipid. Thus, synthesis of a desired oligosaccharidehas required the use of a different glycosyltransferase for eachdifferent saccharide unit being transferred.

[0009] More specifically, such glycosyltransferases have only providedfor the transfer of a single saccharide unit, specific for theglycosyltransferases. For example a galactosyltransferase would transferonly galactose, a glucosyltransferase would transfer only glucose, anN-acetylglucosamine and a sialyl transferase would transfer only sialicacid.

[0010] However, the lack of generality of glycosyltransferase, makes itnecessary to use a different glycosyltransferase for every differentsugar donor being transferred. As the usefulness of oligosaccharidecompounds expands, the ability to transfer more than one sugar donorwould provide a tremendous advantage, by decreasing the number ofglycosyltransferases necessary to form necessary glycosidic bonds.

[0011] In addition, a glycosyltransferase which transferred at least twodifferent sugar donors would be advantageous in synthesizing twoglycosidic bonds of at least a trisaccharide, using the sameglycosyltransferase.

[0012] A locus involved in the biosynthesis of gonococcallipooligosaccharide (LOS) has been reported as being cloned from thegonococcal strain F62 (Gotschlich J. Exp. Med (1994) 180, 2181-2190).Five genes lgtA, lgtB, lgtC, lgtD and lgtE are reported, and based ondeletion experiments, activities are postulated, as encoding forglycosyltransferases. Due to the uncertainty caused by the nature of thedeletion experiments, the exact activity of the proteins encoded by eachof the genes was not ascertained and some of the genes are onlysuggested as being responsible for one or another activity, in thealternative. The gene lgtA is suggested as most likely to code for aGlcNAc transferase.

[0013] The transfer of more than one different saccharide moietie, by apolyglycosyltransferase has heretofore been unreported.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to a method of transferring atleast two saccharide units with a polyglycosyltransferase, apolyglycosyltransferase and nucleic acids encoding apolyglycosyltransferase.

[0015] Accordingly, in one aspect, the invention is directed to a methodof transferring at least two saccharide units with apolyglycosyltransferase.

[0016] Accordingly, another aspect of the invention is directed to amethod of transferring at least two saccharide units with apolyglycosyltransferase, which transfers both GlcNAc and GalNAc, fromthe corresponding sugar nucleotides, to a sugar acceptor.

[0017] According to another aspect of the invention, apolyglycosyltransferase is obtained from a bacteria of the genusNeisseria, Escherichia or Pseudomonas.

[0018] According to another aspect of the invention, is directed to amethod of making at least two oligosaccharide compounds, from the sameacceptor, with a polyglycosyltransferase.

[0019] Accordingly, another aspect of the invention is directed to amethod of making at least two oligosaccharide compounds, from the sameacceptor, with a polyglycosyltransferase, which transfers both GlcNAcand Ga1NAc, from the corresponding sugar nucleotides, to the sugaracceptor.

[0020] According to another embodiment of the present invention is amethod of transferring an N-acetylgalactosamine using aglycosyltransferase of SEQ ID NO:3

[0021] In specific embodiments, the invention relates to a nucleic acidthat has a nucleotide sequence which encodes for polypeptide sequenceshown in SEQ ID NO: 3.

[0022] The functionally active polyglycosyltransferase of the inventionis characterized by catalyzing both the addition of GalNAc β1-3 to Galand the addition of G1cNAc β1-3 to Gal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1: provides the amino acid sequence of apolyglycosyltransferase of SEQ ID NO. 3.

[0024]FIG. 2: provides the polynucleotide sequence of a LOS encodinggene isolated from N. gonorrhoeae, of which nucleotides 445-1488 encodefor a polyglycosyltransferase

DETAILED DESCRIPTION OF THE INVENTION

[0025] As disclosed above, the present invention provides for a methodof transferring at least two saccharide units with apolyglycosyltransferase, a gene encoding for a polyglycosyltransferase,and a polyglycosyltransferase. The polyglycosyltransferases of theinvention can be used for in vitro biosynthesis of variousoligosaccharides, such as the core oligosaccharide of the human bloodgroup antigens, i.e., lacto-N-neotetraose.

[0026] Cloning and expression of a polyglycosyltransferase of theinvention can be accomplished using standard techniques, as disclosedherein. Such a polyglycosyltransferase is useful for biosynthesis ofoligosaccharides in vitro, or alternatively genes encoding such apolyglycosyltransferase can be transfected into cells, e.g., yeast cellsor eukaryotic cells, to provide for alternative glycosylation ofproteins and lipids.

[0027] The instant invention is based, in part, on the discovery that apolyglycosyltransferase isolated from Neisseria gonorrhoeae is capableof transferring both GlcNAc β1-3 to Gal and GalNAc β1-3 to Gal, from thecorresponding sugar nucleotides.

[0028] A protein, glycosyltransferase activity, is reported byGotschlich et al U.S. Ser. No. 08/312,387 filed on Sep. 26, 1994, bycloning of a locus involved in the biosynthesis of gonococcal LOS,strain F62. The protein sequence identified as SEQ ID NO: 3, a 348 aminoacid protein, has now been discovered to have a polyglycosyltransferaseactivity. More specifically, the protein sequence identified as SEQ IDNO: 3 has been discovered to transfer both GlcNAc β1-3 to Gal and Ga1NAcβ1-3 to Gal, from the corresponding sugar nucleotides.

[0029] In addition to the protein sequence SEQ ID NO: 3 and nucleic acidsequences for encoding them reported in U.S. Ser. No. 08/312,387, newpolyglycosyltransferases have been discovered which transfer twodifferent sugar units. This protein is similar to the protein of SEQ ID:3 with the deletion of one or two of the five glycine units occurringbetween amino acid nos 86-90 of lgtA. In addition, an additional aminoacid sequence -Tyr-Ser-Arg-Asp-Ser-Ser can be appended to the carboxyterminus of Ile (amino acid no 348) of SEQ ID NO: 3, while retaining thepolyglycosyltransferase activity.

[0030] A polynucleotide sequence encoding for a polyglycosyltransferaseis similar to the sequence of nucleic acids no 445 to 1488 of an LOSisolated from N. gonorrhoeae (see FIG. 2) in which three or six of theguanine units occurring between nucleic acids no 700 to 715 have beendeleted.

[0031] Another polynucleotide sequence is similar to the sequence ofnucleic acids no 445 to 1488 of an LOS isolated from N. gonorrhoeae (seeFIG. 2), in which nucleic acids sufficient to encode the amino acidsequence -Tyr-Ser-Arg-Asp-Ser-Ser, can be appended to nucleic acid 1488.

[0032] Abbreviations used throughout this specification include:Lipopolysaccharide, LPS; Lipooligosaccharide, LOS; N-Acetyl-neuraminicacid cytidine mono phosphate, CMP-NANA; wild type, wt; Gal, galactose;Glc, glucose; NAc, N-acetyl (e.g., GalNAc or GlcNAc).

Isolation of Genes for Poly Glycosyltransferases

[0033] Any Neisseria bacterial cell can potentially serve as the nucleicacid source for the molecular cloning of a polyglycosyltransferase gene.In a specific embodiment, infra, the genes are isolated from Neisseriagonorrhoeae. The DNA may be obtained by standard procedures known in theart from cloned DNA (e.g., a DNA “library'), by chemical synthesis, bycDNA cloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired cell (See, for example, Sambrook et al., 1989,supra Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U.K. Vol. 1, II). For example, a N. gonorrhoeaegenomic DNA can be digested with a restriction endonuclease orendonucleases, e.g., Sau3A, into a phage vector digested with arestriction endonuclease or endonucleases, e.g., BamHI/EcoRI, forcreation of a phage genomic library. Whatever the source, the geneshould be molecularly cloned into a suitable vector for propagation ofthe gene.

[0034] In the molecular cloning of the gene from genomic DNA, DNAfragments are generated, some of which will encode the desired gene. TheDNA may be cleaved at specific sites using; various restriction enzymes.Alternatively, one may use DNAse the presence of manganese to fragmentthe DNA, or the DNA can be physically sheared, as for example, bysonication. The linear DNA fragments can then be separated according tosize by standard techniques, including but not limited to, agarose andpolyacrylamide gel electrophoresis and column chromatography.

[0035] Once the DNA fragments are generated, identification of thespecific DNA fragment containing the desired polyglycosyltransferasegene may be accomplished in a number of ways. For example, the generatedDNA fragments may be screened by nucleic acid hybridization to thelabeled probe synthesized with a sequence as disclosed herein (Bentonand Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc.Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantialhomology to the probe will hybridize.

[0036] Suitable probes can be generated by PCR using random primers. Inparticular a probe which will hybridize to the polynucleotide sequenceencoding for a four or five glycine residue (i.e. a twelve or fifteenguanine residue) would be a suitable probe for apolyglycosyltransferase.

[0037] As described above, the presence of the gene may be detected byassays based on the physical, chemical, or immunological properties ofits expressed product. For example DNA clones that produce a proteinthat, e.g., has similar or identical electrophoretic migration,isoelectric focusing behavior, proteolytic digestion maps, proteolyticactivity, or functional properties, in particularpolyglycosyltransferase activity, the ability of apolyglycosyltransferase protein to mediate transfer of two differentsaccharide units to an acceptor molecule.

[0038] Alternatives to isolating a polyglycosyltransferase genomic DNAinclude, but are not limited to, chemically synthesizing the genesequence itself from a known sequence that encodes apolyglycosyltransferase. In another embodiment, DNA for apolyglycosyltransferase gene can be isolated by PCR usingoligonucleotide primers designed from the nucleotide sequences disclosedherein. Other methods are possible and within the scope of theinvention.

[0039] The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. In a specific aspect of theinvention, the polyglycosyltransferase coding sequence is inserted in anE. coli cloning vector. Other examples of vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors,pmal-c, pFLAG, etc. The insertion into a cloning vector can, forexample, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini, theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In specific embodiment, PCR primers containing such linkersites can be used to amplify the DNA for cloning. Recombinant moleculescan be introduced into host cells via transformation, transfection,infection, electroporation, etc., so that many copies of the genesequence are generated.

[0040] Transformation of host cells with recombinant DNA molecules thatincorporate the isolated polyglycosyltransferase gene or synthesized DNAsequence enables generation of multiple copies of the gene. Thus, thegene may be obtained in large quantities by growing transformants,isolating the recombinant DNA molecules from the transformants and, whennecessary, retrieving the inserted gene from the isolated recombinantDNA.

[0041] The present invention also relates to vectors containing genesencoding truncated forms of the enzyme (fragments) and derivatives ofpolyglycosyltransferases that have the same functional activity as apolyglycosyltransferase. The production and use of fragments andderivatives related to polyglycosyltransferases are within the scope ofthe present invention. In a specific embodiment, the fragment orderivative is functionally active, i.e., capable of mediating transferof two sugar donors to a acceptor moieties.

[0042] Truncated fragments of the polyglycosyltransferases can beprepared by eliminating N-terminal, C-terminal, or internal regions ofthe protein that are not required for functional activity. Usually, suchportions that are eliminated will include only a few, e.g., between 1and 5, amino acid residues, but larger segments may be removed.

[0043] Chimeric molecules, e.g., fusion proteins, containing all or afunctionally active portion of a polyglycosyltransferase of theinvention joined to another protein are also envisioned. Apolyglycosyltransferase fusion protein comprises at least a functionallyactive portion of a non-glycosyltransferase protein joined via a peptidebond to at least a functionally active portion of apolyglycosyltransferase polypeptide. The non-glycosyltransferasesequences can be amino- or carboxy-terminal to thepolyglycosyltransferase sequences. Expression of a fusion protein canresult in an enzymatically inactive polyglycosyltransferase fusionprotein. A recombinant DNA molecule encoding such a fusion proteincomprises a sequence encoding at least a functionally active portion ofa non-glycosyltransferase protein joined in-frame to thepolyglycosyltransferase coding sequence, and preferably encodes acleavage site for a specific protease, e.g., thrombin or Factor Xa,preferably at the polyglycosyltransferase-non-glycosyltransferasejuncture. In a specific embodiment, the fusion protein may be expressedin Escherichia coli.

[0044] In particular, polyglycosyltransferase derivatives can be made byaltering encoding nucleic acid sequences by substitutions, additions ordeletions that provide for functionally equivalent molecules. Due to thedegeneracy of nucleotide coding sequences, other DNA sequences whichencode substantially the same amino acid sequence as anpolyglycosyltransferase gene may be used in the practice of the presentinvention. These include but are not limited to nucleotide sequencescomprising all or portions of polyglycosyltransferase genes that arealtered by the substitution of different codons that encode the sameamino acid residue within the sequence, thus producing a silent change.Likewise, the polyglycosyltransferase derivatives of the inventioninclude, but are not limited to, those containing, as a primary aminoacid sequence, all or part of the amino acid sequence of apolyglycosyltransferase including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a conservative amino acid substitution.For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0045] The genes encoding polyglycosyltransferase derivatives andanalogs of the invention can be produced by various methods known in theart (e.g., Sambrook et al., 1989, supra). The sequence can be cleaved atappropriate sites with restriction endonuclease(s), followed by furtherenzymatic modification if desired, isolated, and ligated in vitro. Inthe production of the gene encoding a derivative or analog ofpolyglycosyltransferase, care should be taken to ensure that themodified gene remains within the same translational reading frame as thepolyglycosyltransferase gene, uninterrupted by translational stopsignals, in the gene region where the desired activity is encoded.

[0046] Additionally, the polyglycosyltransferase nucleic acid sequencecan be mutated in vitro or in vivo, to create and/or destroytranslation, initiation, and/or termination sequences, or to createvariations in coding regions and/or form new restriction endonucleasesites or destroy preexisting ones, to facilitate further in vitromodification. Any technique for mutagenesis known in the art can beused, including but not limited to, in vitro site-directed mutagenesis(Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551; Zoller andSmith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177;Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use ofTABO linkers (Pharmacia), etc. PCR techniques are preferred for sitedirected mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA”, inPCR Technology: Principles and Applications for “DNA Amplification, H.Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

[0047] While a polyglycosyltransferase has been isolated from a bacteriaof Neisseria gonorrhoeae, polyglycosyltransferases can also be isolatedfrom other bacterial species of Neisseria. Exemplary Neisseria bacterialsources include N. animalis (ATCC 19573), N. canis (ATCC 14687), N.cinerea (ATCC 14685), N. cuniculi (ATCC 14688), N. denitrificans (ATCC14686), N. elongata (ATCC 25295), N. elongata subsp glycolytica (ATCC29315), N. elongata subsp. nitroreducens (ATCC 49377), N. flavescens(ATCC 13115), N. gonorrhoeae (ATCC 33084), N. lactamica (ATCC 23970), N.macaca (ATCC 33926), N. meningitidis, N. mucosa (ATCC 19695), N. mucosasubsp. eidelbergensis (ATCC 25998), N. polysaccharea (ATCC 43768), N.sicca (ATCC 29256) and N. subflava (ATCC 49275). In additionpolyglycosyltransferases can be isolated from Branhamella catarrhalis,Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa andPseudomonas cepacia.

Expression of a Polyglycosyltransferase

[0048] The gene coding for a polyglycosyltransferase, or a functionallyactive fragment or other derivative thereof, can be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. An expression vector also preferably includes areplication origin. The necessary transcriptional and translationalsignals can also be supplied by the native polyglycosyltransferase geneand/or its flanking regions. A variety of host-vector systems may beutilized to express the protein-coding sequence. Preferably, however, abacterial expression system is used to provide for high level expressionof the protein with a higher probability of the native conformation.Potential host-vector systems include but are not limited to mammaliancell systems infected with virus (e.g., vaccine virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

[0049] Preferably, the periplasmic form of the polyglycosyltransferase(containing a signal sequence) is produced for export of the protein tothe Escherichia coli periplasm or in an expression system based onBacillus subtillis.

[0050] Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).

[0051] Expression of nucleic acid sequence encoding apolyglycosyltransferase or peptide fragment may be regulated by a secondnucleic acid sequence so that the polyglycosyltransferase or peptide isexpressed in a host transformed with the recombinant DNA molecule. Forexample, expression of a polyglycosyltransferase may be controlled byany promoter/enhancer element known in the art, but these regulatoryelements must be functional in the host selected for expression. Forexpression in bacteria, bacterial promoters are required. Eukaryoticviral or eukaryotic promoters, including tissue specific promoters, arepreferred when a vector containing a polyglycosyltransferase gene isinjected directly into a subject for transient expression, resulting inheterologous protection against bacterial infection, as described indetail below. Promoters which may be used to controlpolyglycosyltransferase gene expression include, but are not limited to,the SV40 early promoter region (Benoist and Chambon, 198 1, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the O-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94; and the like

[0052] Expression vectors containing polyglycosyltransferase geneinserts can be identified by four general approaches: (a) PCRamplification of the desired plasmid DNA or specific mRNA, (b) nucleicacid hybridization, (c) presence or absence of “marker” gene functions,and (d) expression of inserted sequences. In the first approach, thenucleic acids can be amplified by PCR with incorporation ofradionucleotides or stained with ethidiuin bromide to provide fordetection of the amplified product. In the second approach, the presenceof a foreign gene inserted in an expression vector can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to an inserted polyglycosyltransferase gene. In the thirdapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., 3-galactosidase activity, PhoA activity, thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, etc.) caused by the insertionof foreign genes in the vector. If the polyglycosyltransferase gene isinserted within the marker gene sequence of the vector, recombinantscontaining the polyglycosyltransferase insert can be identified by theabsence of the marker gene function. In the fourth approach, recombinantexpression vectors can be identified by assaying for the activity of thepolyglycosyltransferase gene product expressed by the recombinant. Suchassays can be based, for example, on the physical or functionalproperties of the polyglycosyltransferase gene product in in vitro assaysystems, e.g., polyglycosyltransferase activity. Once a suitable hostsystem and growth conditions are established, recombinant expressionvectors can be propagated and prepared in quantity.

[0053] Biosynthesis of Oligosaccharides

[0054] The polyglycosyltransferase of the present invention can be usedin the biosynthesis of oligosaccharides. The polyglycosyltransferases ofthe invention are capable of stereospecific conjugation of two specificactivated saccharide unit to specific acceptor molecules. Such activatedsaccharides generally consist of uridine or guanosine diphosphate andcytidine monophosphate derivatives of the saccharides, in which thenucleoside mono- and diphosphate serves as a leaving group. Thus, theactivated saccharide may be a saccharide-UDP, a saccharide-GDP, or asaccharide-CMP. In specific embodiments, the activated saccharide isUDP-G1cNAc, UDP-Ga1NAc, or UDP-Gal.

[0055] Within the context of the claimed invention, two differentsaccharide units means saccharides which differ in structure and/orstereochemistry at a position other than C₁ and accordingly the pyranoseand furanose of the same carbon backbone are considered to be the samesaccharide unit, while glucose and galactose (i.e. C₄ isomers) areconsidered different.

[0056] A glycosyltransferase typically has a catalytic activity of fromabout 1 to 250 turnovers/sec in order to be considered to possess aspecific glycosyltransferase activity.

[0057] Accordingly each individual glycosyltransferase activity of thepolyglycosyltransferase of the present invention, is within the range offrom 1 to 250 turnovers/sec, preferably from 5 to 100 turnovers/sec,more preferably from 10 to 30 turnovers/sec.

[0058] In addition to absolute glycosyltransferase activity, therelative rates of each of the identified glycosyltransferase activities,in the polyglycosyltransferase, has relative activities of from 0.1 to10 times, preferably from 0.2 to 5 times, more preferably from 0.5 to 2times and most preferably from 0.8 to 1.5, the rate of any one of theother glycosyltransferase activities.

[0059] The term “acceptor moiety” as used herein refers to the moleculesto which the polyglycosyltransferase transfers activated sugars.

[0060] For the synthesis of an oligosaccharide, apolyglycosyltransferase is contacted with an appropriate activatedsaccharide and an appropriate acceptor moiety under conditions effectiveto transfer and covalently bond the saccharide to the acceptor molecule.Conditions of time, temperature, and pH appropriate and optimal for aparticular saccharine unit transfer can be determined through routinetesting; generally, physiological conditions will be acceptable. Certainco-reagents may also be desirable; for example, it may be more effectiveto contact the polyglycosyltransferase with the activated saccharide andthe acceptor moiety in the presence of a divalent cation.

[0061] According to the invention, the polyglycosyltransferase enzymescan be covalently or non-covalently immobilized on a solid phase supportsuch as SEPHADEX, SEPHAROSE, or poly(acrylamide-co-N-acryloxysucciimide)(PAN) resin. A specific reaction can be performed in an isolatedreaction solution, with facile separation of the solid phase enzyme fromthe reaction products. Immobilization of the enzyme also allows for acontinuous biosynthetic stream, with the specificpolyglycosyltransferases attached to a solid support, with the supportsarranged randomly or in distinct zones in the specified order in acolumn, with passage of the reaction solution through the column andelution of the desired oligosaccharide at the end. An efficient methodfor attaching the polyglycosyltransferase to a solid support and usingsuch immobilized polyglycosyltransferases is described in U.S. Pat. No.5,180,674, issued Jan. 19, 1993 to Roth, which is specificallyincorporated herein by reference in its entirety.

[0062] An oligosaccharide, e.g., a disaccharide, prepared using apolyglycosyltransferase of the present invention can serve as anacceptor moiety for further synthesis, either using otherpolyglycosyltransferases of the invention, or glycosyltransferases knownin the art (see, e.g., Roth, U.S. Pat. No. 5,180,674).

[0063] Alternatively, the polyglycosyltransferases of the presentinvention can be used to prepareGalNAcβ1-3-Galβ1-4-GlcNAcβ1-3-Galβ1-4-Glc orGalNAcβ1-3-Galβ1-4-GlcNAcβ1-3-Galβ1-4-GlcNAc from lactose or lactosaminerespectively, in which a polyglycosyltransferase is used to synthesizeboth the GlcNAc β1-3-Gal and GalNAc β1-3 Gal linkages.

[0064] Accordingly, a method for preparing an oligosaccharide having thestructure GalNAcβ1-3-Galβ1-4-G1cNAc β1-3-Galβ1-4-Glc comprisessequentially performing the steps of:

[0065] a) contacting a reaction mixture comprising an activated GlcNAc(such as UDP-GlcNAc) to lactose with a polyglycosyltransferase having anamino acid sequence of SEQ ID NO:3, or a functionally active fragmentthereof;

[0066] b) contacting a reaction mixture comprising an activated Gal(i.e. UDP-Gal) to the acceptor moiety comprising aGlcNAcβ1-3-Galβ1-4-Glc residue in the presence of aβ1-4-galactosyltransferase; and

[0067] c) contacting a reaction mixture comprising an activated GalNAc(i.e. UDP-GalNAc) to the acceptor moiety comprising aGalβ1-4-GlcNAcβ1-3-Galβ1-4-Glc residue in the presence of thepolyglycosyltransferase of step a).

[0068] A suitable β1-4 galactosyltransferase can be isolated from bovinemilk.

[0069] Oligosaccharide synthesis, using a polyglycosyltransferase isgenerally conducted at a temperature of from 15 to 38° C., preferablyfrom 20 to 25° C. While enzymatic activity is comparable at 25° C. and37° C., the polyglycosyltransferase stability is greater at 25° C.

[0070] In a preferred embodiment polyglycosyltransferase activity isobserved in the absence of α-lactalbumin.

[0071] In a preferred embodiment polyglycosyltransferase activity isobserved at the same pH, more preferably at pH 6.5 to 7.5.

[0072] In a preferred embodiment polyglycosyltransferase activity isobserved at the same temperature.

[0073] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

EXAMPLE 1

[0074] Synthesis of GalNAcβ1-3-Galβ1-4-G1cNAcβ1-3-Ga1β1-4-Glc:

[0075] Lactose was contacted with UDP-N-acetylglucosamine and aβ-galactoside β1-3 N-acetylglucosaminyl transferase of SEQ ID NO: 3, ina 0.5 M HEPES buffered aqueous solution at 25° C. The producttrisaccharide was then contacted with UDP-Gal and aβ-N-acetylglucosaminoside β1-4 Galactosyltransferase isolated frombovine milk, in a 0.05 M HEPES buffered aqueous solution at 37° C. Theproduct tetrasaccharide was then contacted withUDP-N-acetylgalactosamine and a β-galactoside β1-3N-acetylgalactosaminyl transferase of SEQ ID NO: 3, in a 0.05 M HEPESbuffered aqueous solution at 25° C. The title pentasaccharide wasisolated by conventional methods.

[0076] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedtherein.

1 8 1 5859 DNA Neisseria gonorrhoeae 1 ctgcaggccg tcgccgtatt caaacaactgcccgaagccg ccgcgctcgc cgccgccaac 60 aaacgcgtgc aaaacctgct gaaaaaagccgatgccgcgt tgggcgaagt caatgaaagc 120 ctgctgcaac aggacgaaga aaaagccctgtacgctgccg cgcaaggttt gcagccgaaa 180 attgccgccg ccgtcgccga aggcaatttccgaaccgcct tgtccgaact ggcttccgtc 240 aagccgcagg ttgatgcctt cttcgacggcgtgatggtga tggcggaaga tgccgccgta 300 aaacaaaacc gcctgaacct gctgaaccgcttggcagagc agatgaacgc ggtggccgac 360 atcgcgcttt tgggcgagta accgttgtacagtccaaatg ccgtctgaag ccttcaggcg 420 gcatcaaatt atcgggagag taaattgcagcctttagtca gcgtattgat ttgcgcctac 480 aacgtagaaa aatattttgc ccaatcattagccgccgtcg tgaatcagac ttggcgcaac 540 ttggatattt tgattgtcga tgacggctcgacagacggca cacttgccat tgccaaggat 600 tttcaaaagc gggacagccg tatcaaaatccttgcacaag ctcaaaattc cggcctgatt 660 ccctctttaa acatcgggct ggacgaattggcaaagtcgg gggggggggg gggggaatat 720 attgcgcgca ccgatgccga cgatattgcctcccccggct ggattgagaa aatcgtgggc 780 gagatggaaa aagaccgcag catcattgcgatgggcgcgt ggctggaagt tttgtcggaa 840 gaaaaggacg gcaaccggct ggcgcggcaccacaaacacg gcaaaatttg gaaaaagccg 900 acccggcacg aagacatcgc cgcctttttccctttcggca accccataca caacaacacg 960 atgattatgc ggcgcagcgt cattgacggcggtttgcgtt acgacaccga gcgggattgg 1020 gcggaagatt accaattttg gtacgatgtcagcaaattgg gcaggctggc ttattatccc 1080 gaagccttgg tcaaataccg ccttcacgccaatcaggttt catccaaaca cagcgtccgc 1140 caacacgaaa tcgcgcaagg catccaaaaaaccgccagaa acgatttttt gcagtctatg 1200 ggttttaaaa cccggttcga cagcctagaataccgccaaa caaaagcagc ggcgtatgaa 1260 ctgccggaga aggatttgcc ggaagaagattttgaacgcg cccgccggtt tttgtaccaa 1320 tgcttcaaac ggacggacac gccgccctccggcgcgtggc tggatttcgc ggcagacggc 1380 aggatgaggc ggctgtttac cttgaggcaatacttcggca ttttgtaccg gctgattaaa 1440 aaccgccggc aggcgcggtc ggattcggcagggaaagaac aggagattta atgcaaaacc 1500 acgttatcag cttggcttcc gccgcagaacgcagggcgca cattgccgca accttcggca 1560 gtcgcggcat cccgttccag tttttcgacgcactgatgcc gtctgaaagg ctggaacggg 1620 caatggcgga actcgtcccc ggcttgtcggcgcaccccta tttgagcgga gtggaaaaag 1680 cctgctttat gagccacgcc gtattgtgggaacaggcatt ggacgaaggc gtaccgtata 1740 tcgccgtatt tgaagatgat gtcttactcggcgaaggcgc ggagcagttc cttgccgaag 1800 atacttggct gcaagaacgc tttgaccccgattccgcctt tgtcgtccgc ttggaaacga 1860 tgtttatgca cgtcctgacc tcgccctccggcgtggcgga ctacggcggg cgcgcctttc 1920 cgcttttgga aagcgaacac tgcgggacggcgggctatat tatttcccga aaggcgatgc 1980 gttttttctt ggacaggttt gccgttttgccgcccgaacg cctgcaccct gtcgatttga 2040 tgatgttcgg caaccctgac gacagggaaggaatgccggt ttgccagctc aatcccgcct 2100 tgtgcgccca agagctgcat tatgccaagtttcacgacca aaacagcgca ttgggcagcc 2160 tgatcgaaca tgaccgccgc ctgaaccgcaaacagcaatg gcgcgattcc cccgccaaca 2220 cattcaaaca ccgcctgatc cgcgccttgaccaaaatcgg cagggaaagg gaaaaacgcc 2280 ggcaaaggcg cgaacagtta atcggcaagattattgtgcc tttccaataa aaggagaaaa 2340 gatggacatc gtatttgcgg cagacgacaactatgccgcc tacctttgcg ttgcggcaaa 2400 aagcgtggaa gcggcccatc ccgatacggaaatcaggttc cacgtcctcg atgccggcat 2460 cagtgaggaa aaccgggcgg cggttgccgccaatttgcgg ggggggggta atatccgctt 2520 tatagacgta aaccccgaag atttcgccggcttcccctta aacatcaggc acatttccat 2580 tacgacttat gcccgcctga aattgggcgaatacattgcc gattgcgaca aagtcctgta 2640 tctggatacg gacgtattgg tcagggacggcctgaagccc ttatgggata ccgatttggg 2700 cggtaactgg gtcggcgcgt gcatcgatttgtttgtcgaa aggcaggaag gatacaaaca 2760 aaaaatcggt atggcggacg gagaatattatttcaatgcc ggcgtattgc tgatcaacct 2820 gaaaaagtgg cggcggcacg atattttcaaaatgtcctgc gaatgggtgg aacaatacaa 2880 ggacgtgatg caatatcagg atcaggacattttgaacggg ctgtttaaag gcggggtgtg 2940 ttatgcgaac agccgtttca actttatgccgaccaattat gcctttatgg cgaacgggtt 3000 tgcgtcccgc cataccgacc cgctttacctcgaccgtacc aatacggcga tgcccgtcgc 3060 cgtcagccat tattgcggct cggcaaagccgtggcacagg gactgcaccg tttggggtgc 3120 ggaacgtttc acagagttgg ccggcagcctgacgaccgtt cccgaagaat ggcgcggcaa 3180 acttgccgtc ccgccgacaa agtgtatgcttcaaagatgg cgcaaaaagc tgtctgccag 3240 attcttacgc aagatttatt gacggggcaggccgtctgaa gccttcagac ggcatcggac 3300 gtatcggaaa ggagaaacgg attgcagcctttagtcagcg tattgatttg cgcctacaac 3360 gcagaaaaat attttgccca atcattggccgccgtagtgg ggcagacttg gcgcaacttg 3420 gatattttga ttgtcgatga cggctcgacggacggcacgc ccgccattgc ccggcatttc 3480 caagaacagg acggcaggat caggataatttccaatcccc gcaatttggg ctttatcgcc 3540 tctttaaaca tcgggctgga cgaattggcaaagtcggggg ggggggaata tattgcgcgc 3600 accgatgccg acgatattgc ctcccccggctggattgaga aaatcgtggg cgagatggaa 3660 aaagaccgca gcatcattgc gatgggcgcgtggttggaag ttttgtcgga agaaaacaat 3720 aaaagcgtgc ttgccgccat tgcccgaaacggcgcaattt gggacaaacc gacccggcat 3780 gaagacattg tcgccgtttt ccctttcggcaaccccatac acaacaacac gatgattatg 3840 aggcgcagcg tcattgacgg cggtttgcggttcgatccag cctatatcca cgccgaagac 3900 tataagtttt ggtacgaagc cggcaaactgggcaggctgg cttattatcc cgaagccttg 3960 gtcaaatacc gcttccatca agaccagacttcttccaaat acaacctgca acagcgcagg 4020 acggcgtgga aaatcaaaga agaaatcagggcggggtatt ggaaggcggc aggcatagcc 4080 gtcggggcgg actgcctgaa ttacgggcttttgaaatcaa cggcatatgc gttgtacgaa 4140 aaagccttgt ccggacagga tatcggatgcctccgcctgt tcctgtacga atatttcttg 4200 tcgttggaaa agtattcttt gaccgatttgctggatttct tgacagaccg cgtgatgagg 4260 aagctgtttg ccgcaccgca atataggaaaatcctgaaaa aaatgttacg cccttggaaa 4320 taccgcagct attgaaaccg aacaggataaatcatgcaaa accacgttat cagcttggct 4380 tccgccgcag agcgcagggc gcacattgccgataccttcg gcagtcgcgg catcccgttc 4440 cagtttttcg acgcactgat gccgtctgaaaggctggaac aggcgatggc ggaactcgtc 4500 cccggcttgt cggcgcaccc ctatttgagcggagtggaaa aagcctgctt tatgagccac 4560 gccgtattgt gggaacaggc gttggatgaaggtctgccgt atatcgccgt atttgaggac 4620 gacgttttac tcggcgaagg cgcggagcagttccttgccg aagatacttg gttggaagag 4680 cgttttgaca aggattccgc ctttatcgtccgtttggaaa cgatgtttgc gaaagttatt 4740 gtcagaccgg ataaagtcct gaattatgaaaaccggtcat ttcctttgct ggagagcgaa 4800 cattgtggga cggctggcta tatcatttcgcgtgaggcga tgcggttttt cttggacagg 4860 tttgccgttt tgccgccaga gcggattaaagcggtagatt tgatgatgtt tacttatttc 4920 tttgataagg aggggatgcc tgtttatcaggttagtcccg ccttatgtac ccaagaattg 4980 cattatgcca agtttctcag tcaaaacagtatgttgggta gcgatttgga aaaagatagg 5040 gaacaaggaa gaagacaccg ccgttcgttgaaggtgatgt ttgacttgaa gcgtgctttg 5100 ggtaaattcg gtagggaaaa gaagaaaagaatggagcgtc aaaggcaggc ggagcttgag 5160 aaagtttacg gcaggcgggt catattgttcaaatagtttg tgtaaaatat aggggattaa 5220 aatcagaaat ggacacactg tcattcccgcgcaggcggga atctaggtct ttaaacttcg 5280 gttttttccg ataaattctt gccgcattaaaattccagat tcccgctttc gcggggatga 5340 cggcgggggg attgttgctt tttcggataaaatcccgtgt tttttcatct gctaggtaaa 5400 atcgccccaa agcgtctgca tcgcggcgatggcggcgagt ggggcggttt ctgtgcgtaa 5460 aatccgtttt ccgagtgtaa ccgcctgaaagccggcttca aatgcctgtt gttcttcctg 5520 ttctgtccag ccgccttcgg gcccgaccataaagacgatt gcgccggacg ggtggcggat 5580 gtcgccgagt ttgcaggcgc ggttgatgctcataatcagc ttggtgtttt cagacggcat 5640 tttgtcgagt gcttcacggt agccgatgatgggcagtacg gggggaacgg tgttcctgcc 5700 gctttgttcg cacgcggaga tgacgatttcctgccagcgt gcgaggcgtt tggcggcgcg 5760 ttctccgtcg aggcggacga tgcagcgttcgctgatgacg ggctgtatgg cggttacgcc 5820 gagttcgacg cttttttgca gggtgaaatccatgcgatc 5859 2 126 PRT Neisseria gonorrhoeae 2 Leu Gln Ala Val Ala ValPhe Lys Gln Leu Pro Glu Ala Ala Ala Leu 1 5 10 15 Ala Ala Ala Asn LysArg Val Gln Asn Leu Leu Lys Lys Ala Asp Ala 20 25 30 Ala Leu Gly Glu ValAsn Glu Ser Leu Leu Gln Gln Asp Glu Glu Lys 35 40 45 Ala Leu Tyr Ala AlaAla Gln Gly Leu Gln Pro Lys Ile Ala Ala Ala 50 55 60 Val Ala Glu Gly AsnPhe Arg Thr Ala Leu Ser Glu Leu Ala Ser Val 65 70 75 80 Lys Pro Gln ValAsp Ala Phe Phe Asp Gly Val Met Val Met Ala Glu 85 90 95 Asp Ala Ala ValLys Gln Asn Arg Leu Asn Leu Leu Asn Arg Leu Ala 100 105 110 Glu Gln MetAsn Ala Val Ala Asp Ile Ala Leu Leu Gly Glu 115 120 125 3 348 PRTNeisseria gonorrhoeae 3 Leu Gln Pro Leu Val Ser Val Leu Ile Cys Ala TyrAsn Val Glu Lys 1 5 10 15 Tyr Phe Ala Gln Ser Leu Ala Ala Val Val AsnGln Thr Trp Arg Asn 20 25 30 Leu Asp Ile Leu Ile Val Asp Asp Gly Ser ThrAsp Gly Thr Leu Ala 35 40 45 Ile Ala Lys Asp Phe Gln Lys Arg Asp Ser ArgIle Lys Ile Leu Ala 50 55 60 Gln Ala Gln Asn Ser Gly Leu Ile Pro Ser LeuAsn Ile Gly Leu Asp 65 70 75 80 Glu Leu Ala Lys Ser Gly Gly Gly Gly GlyGlu Tyr Ile Ala Arg Thr 85 90 95 Asp Ala Asp Asp Ile Ala Ser Pro Gly TrpIle Glu Lys Ile Val Gly 100 105 110 Glu Met Glu Lys Asp Arg Ser Ile IleAla Met Gly Ala Trp Leu Glu 115 120 125 Val Leu Ser Glu Glu Lys Asp GlyAsn Arg Leu Ala Arg His His Lys 130 135 140 His Gly Lys Ile Trp Lys LysPro Thr Arg His Glu Asp Ile Ala Ala 145 150 155 160 Phe Phe Pro Phe GlyAsn Pro Ile His Asn Asn Thr Met Ile Met Arg 165 170 175 Arg Ser Val IleAsp Gly Gly Leu Arg Tyr Asp Thr Glu Arg Asp Trp 180 185 190 Ala Glu AspTyr Gln Phe Trp Tyr Asp Val Ser Lys Leu Gly Arg Leu 195 200 205 Ala TyrTyr Pro Glu Ala Leu Val Lys Tyr Arg Leu His Ala Asn Gln 210 215 220 ValSer Ser Lys His Ser Val Arg Gln His Glu Ile Ala Gln Gly Ile 225 230 235240 Gln Lys Thr Ala Arg Asn Asp Phe Leu Gln Ser Met Gly Phe Lys Thr 245250 255 Arg Phe Asp Ser Leu Glu Tyr Arg Gln Thr Lys Ala Ala Ala Tyr Glu260 265 270 Leu Pro Glu Lys Asp Leu Pro Glu Glu Asp Phe Glu Arg Ala ArgArg 275 280 285 Phe Leu Tyr Gln Cys Phe Lys Arg Thr Asp Thr Pro Pro SerGly Ala 290 295 300 Trp Leu Asp Phe Ala Ala Asp Gly Arg Met Arg Arg LeuPhe Thr Leu 305 310 315 320 Arg Gln Tyr Phe Gly Ile Leu Tyr Arg Leu IleLys Asn Arg Arg Gln 325 330 335 Ala Arg Ser Asp Ser Ala Gly Lys Glu GlnGlu Ile 340 345 4 306 PRT Neisseria gonorrhoeae 4 Met Asp Ile Val PheAla Ala Asp Asp Asn Tyr Ala Ala Tyr Leu Cys 1 5 10 15 Val Ala Ala LysSer Val Glu Ala Ala His Pro Asp Thr Glu Ile Arg 20 25 30 Phe His Val LeuAsp Ala Gly Ile Ser Glu Glu Asn Arg Ala Ala Val 35 40 45 Ala Ala Asn LeuArg Gly Gly Gly Asn Ile Arg Phe Ile Asp Val Asn 50 55 60 Pro Glu Asp PheAla Gly Phe Pro Leu Asn Ile Arg His Ile Ser Ile 65 70 75 80 Thr Thr TyrAla Arg Leu Lys Leu Gly Glu Tyr Ile Ala Asp Cys Asp 85 90 95 Lys Val LeuTyr Leu Asp Thr Asp Val Leu Val Arg Asp Gly Leu Lys 100 105 110 Pro LeuTrp Asp Thr Asp Leu Gly Gly Asn Trp Val Gly Ala Cys Ile 115 120 125 AspLeu Phe Val Glu Arg Gln Glu Gly Tyr Lys Gln Lys Ile Gly Met 130 135 140Ala Asp Gly Glu Tyr Tyr Phe Asn Ala Gly Val Leu Leu Ile Asn Leu 145 150155 160 Lys Lys Trp Arg Arg His Asp Ile Phe Lys Met Ser Cys Glu Trp Val165 170 175 Glu Gln Tyr Lys Asp Val Met Gln Tyr Gln Asp Gln Asp Ile LeuAsn 180 185 190 Gly Leu Phe Lys Gly Gly Val Cys Tyr Ala Asn Ser Arg PheAsn Phe 195 200 205 Met Pro Thr Asn Tyr Ala Phe Met Ala Asn Gly Phe AlaSer Arg His 210 215 220 Thr Asp Pro Leu Tyr Leu Asp Arg Thr Asn Thr AlaMet Pro Val Ala 225 230 235 240 Val Ser His Tyr Cys Gly Ser Ala Lys ProTrp His Arg Asp Cys Thr 245 250 255 Val Trp Gly Ala Glu Arg Phe Thr GluLeu Ala Gly Ser Leu Thr Thr 260 265 270 Val Pro Glu Glu Trp Arg Gly LysLeu Ala Val Pro Pro Thr Lys Cys 275 280 285 Met Leu Gln Arg Trp Arg LysLys Leu Ser Ala Arg Phe Leu Arg Lys 290 295 300 Ile Tyr 305 5 337 PRTNeisseria gonorrhoeae 5 Leu Gln Pro Leu Val Ser Val Leu Ile Cys Ala TyrAsn Ala Glu Lys 1 5 10 15 Tyr Phe Ala Gln Ser Leu Ala Ala Val Val GlyGln Thr Trp Arg Asn 20 25 30 Leu Asp Ile Leu Ile Val Asp Asp Gly Ser ThrAsp Gly Thr Pro Ala 35 40 45 Ile Ala Arg His Phe Gln Glu Gln Asp Gly ArgIle Arg Ile Ile Ser 50 55 60 Asn Pro Arg Asn Leu Gly Phe Ile Ala Ser LeuAsn Ile Gly Leu Asp 65 70 75 80 Glu Leu Ala Lys Ser Gly Gly Gly Glu TyrIle Ala Arg Thr Asp Ala 85 90 95 Asp Asp Ile Ala Ser Pro Gly Trp Ile GluLys Ile Val Gly Glu Met 100 105 110 Glu Lys Asp Arg Ser Ile Ile Ala MetGly Ala Trp Leu Glu Val Leu 115 120 125 Ser Glu Glu Asn Asn Lys Ser ValLeu Ala Ala Ile Ala Arg Asn Gly 130 135 140 Ala Ile Trp Asp Lys Pro ThrArg His Glu Asp Ile Val Ala Val Phe 145 150 155 160 Pro Phe Gly Asn ProIle His Asn Asn Thr Met Ile Met Arg Arg Ser 165 170 175 Val Ile Asp GlyGly Leu Arg Phe Asp Pro Ala Tyr Ile His Ala Glu 180 185 190 Asp Tyr LysPhe Trp Tyr Glu Ala Gly Lys Leu Gly Arg Leu Ala Tyr 195 200 205 Tyr ProGlu Ala Leu Val Lys Tyr Arg Phe His Gln Asp Gln Thr Ser 210 215 220 SerLys Tyr Asn Leu Gln Gln Arg Arg Thr Ala Trp Lys Ile Lys Glu 225 230 235240 Glu Ile Arg Ala Gly Tyr Trp Lys Ala Ala Gly Ile Ala Val Gly Ala 245250 255 Asp Cys Leu Asn Tyr Gly Leu Leu Lys Ser Thr Ala Tyr Ala Leu Tyr260 265 270 Glu Lys Ala Leu Ser Gly Gln Asp Ile Gly Cys Leu Arg Leu PheLeu 275 280 285 Tyr Glu Tyr Phe Leu Ser Leu Glu Lys Tyr Ser Leu Thr AspLeu Leu 290 295 300 Asp Phe Leu Thr Asp Arg Val Met Arg Lys Leu Phe AlaAla Pro Gln 305 310 315 320 Tyr Arg Lys Ile Leu Lys Lys Met Leu Arg ProTrp Lys Tyr Arg Ser 325 330 335 Tyr 6 280 PRT Neisseria gonorrhoeae 6Met Gln Asn His Val Ile Ser Leu Ala Ser Ala Ala Glu Arg Arg Ala 1 5 1015 His Ile Ala Asp Thr Phe Gly Ser Arg Gly Ile Pro Phe Gln Phe Phe 20 2530 Asp Ala Leu Met Pro Ser Glu Arg Leu Glu Gln Ala Met Ala Glu Leu 35 4045 Val Pro Gly Leu Ser Ala His Pro Tyr Leu Ser Gly Val Glu Lys Ala 50 5560 Cys Phe Met Ser His Ala Val Leu Trp Glu Gln Ala Leu Asp Glu Gly 65 7075 80 Leu Pro Tyr Ile Ala Val Phe Glu Asp Asp Val Leu Leu Gly Glu Gly 8590 95 Ala Glu Gln Phe Leu Ala Glu Asp Thr Trp Leu Glu Glu Arg Phe Asp100 105 110 Lys Asp Ser Ala Phe Ile Val Arg Leu Glu Thr Met Phe Ala LysVal 115 120 125 Ile Val Arg Pro Asp Lys Val Leu Asn Tyr Glu Asn Arg SerPhe Pro 130 135 140 Leu Leu Glu Ser Glu His Cys Gly Thr Ala Gly Tyr IleIle Ser Arg 145 150 155 160 Glu Ala Met Arg Phe Phe Leu Asp Arg Phe AlaVal Leu Pro Pro Glu 165 170 175 Arg Ile Lys Ala Val Asp Leu Met Met PheThr Tyr Phe Phe Asp Lys 180 185 190 Glu Gly Met Pro Val Tyr Gln Val SerPro Ala Leu Cys Thr Gln Glu 195 200 205 Leu His Tyr Ala Lys Phe Leu SerGln Asn Ser Met Leu Gly Ser Asp 210 215 220 Leu Glu Lys Asp Arg Glu GlnGly Arg Arg His Arg Arg Ser Leu Lys 225 230 235 240 Val Met Phe Asp LeuLys Arg Ala Leu Gly Lys Phe Gly Arg Glu Lys 245 250 255 Lys Lys Arg MetGlu Arg Gln Arg Gln Ala Glu Leu Glu Lys Val Tyr 260 265 270 Gly Arg ArgVal Ile Leu Phe Lys 275 280 7 6 PRT Neisseria gonorrhoeae 7 Tyr Ser ArgAsp Ser Ser 1 5 8 348 PRT Neisseria gonorrhoeae 8 Leu Gln Pro Leu ValSer Val Leu Ile Cys Ala Tyr Asn Val Glu Lys 1 5 10 15 Tyr Phe Ala GlnSer Leu Ala Ala Val Val Asn Gln Thr Trp Arg Asn 20 25 30 Leu Asp Ile LeuIle Val Asp Asp Gly Ser Thr Asp Gly Thr Leu Ala 35 40 45 Ile Ala Lys AspPhe Gln Lys Arg Asp Ser Arg Ile Lys Ile Leu Ala 50 55 60 Gln Ala Gln AsnSer Gly Leu Ile Pro Ser Leu Asn Ile Gly Leu Asp 65 70 75 80 Glu Leu AlaLys Ser Gly Gly Gly Gly Gly Glu Tyr Ile Ala Arg Thr 85 90 95 Asp Ala AspAsp Ile Ala Ser Pro Gly Trp Ile Glu Lys Ile Val Gly 100 105 110 Glu MetGlu Lys Asp Arg Ser Ile Ile Ala Met Gly Ala Trp Leu Glu 115 120 125 ValLeu Ser Glu Glu Lys Asp Gly Asn Arg Leu Ala Arg His His Lys 130 135 140His Gly Lys Ile Trp Lys Lys Pro Thr Arg His Glu Asp Ile Ala Ala 145 150155 160 Phe Phe Pro Phe Gly Asn Pro Ile His Asn Asn Thr Met Ile Met Arg165 170 175 Arg Ser Val Ile Asp Gly Gly Leu Arg Tyr Asp Thr Glu Arg AspTrp 180 185 190 Ala Glu Asp Tyr Gln Phe Trp Tyr Asp Val Ser Lys Leu GlyArg Leu 195 200 205 Ala Tyr Tyr Pro Glu Ala Leu Val Lys Tyr Arg Leu HisAla Asn Gln 210 215 220 Val Ser Ser Lys His Ser Val Arg Gln His Glu IleAla Gln Gly Ile 225 230 235 240 Gln Lys Thr Ala Arg Asn Asp Phe Leu GlnSer Met Gly Phe Lys Thr 245 250 255 Arg Phe Asp Ser Leu Glu Tyr Arg GlnThr Lys Ala Ala Ala Tyr Glu 260 265 270 Leu Pro Glu Lys Asp Leu Pro GluGlu Asp Phe Glu Arg Ala Arg Arg 275 280 285 Phe Leu Tyr Gln Cys Phe LysArg Thr Asp Thr Pro Pro Ser Gly Ala 290 295 300 Trp Leu Asp Phe Ala AlaAsp Gly Arg Met Arg Arg Leu Phe Thr Leu 305 310 315 320 Arg Gln Tyr PheGly Ile Leu Tyr Arg Leu Ile Lys Asn Arg Arg Gln 325 330 335 Ala Arg SerAsp Ser Ala Gly Lys Glu Gln Glu Ile 340 345

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method of transferring at least twosaccharide units with a single enzyme comprising contacting an acceptormoiety and two different sugar donors with a polyglycosyltransferase. 2.The method of claim 1, wherein said two saccharide units areN-acetylglucosamine and N-acetylgalactosamine.
 3. The method of claim 1,wherein said acceptor moiety has a galactose at a non-reducing end. 4.The method of claim 1, wherein said polyglycosyltransferase is isolatedfrom Neisseria.
 5. The method of claim 1, wherein saidpolyglycosyltransferase is isolated from Neisseria gonorrhoeae.
 6. Amethod of transferring N-acetylgalactosamine, to an acceptor moiety,comprising contacting an N-acetylgalactosamine donor and an acceptormoiety with an N-acetylglucosaminyl transferase.
 7. The method of claim6, wherein said N-acetylglucosaminyl transferase is isolated fromNeisseria.
 8. The method of claim 6, wherein said N-acetylglucosaminyltransferase is isolated from Neisseria gonorrhoeae.
 9. The method ofclaim 6, wherein said acceptor moiety has a galactose at a non-reducingend.
 10. The method of claim 8, wherein said Neisseria gonorrhoeae isATCC 33084.